Identification of nucleic acid sequences is a key diagnostic tool in many fields, including medicine, forensics, food production, animal husbandry, and the like, e.g. Jobling et al., Nature Reviews Genetics, 5: 739-751 (2004); Jo et al., Semin. Oncol., 32: 11-23 (2005); Woo et al., J. Clin. Microbiol., 41: 1996-2001 (2003). For example, DNA amplification technologies have found applications in all of these areas, including applications for viral and bacterial detection, viral load monitoring, detection of rare and/or difficult-to-culture pathogens, rapid detection of bio-terror threats, detection of minimal residual disease in cancer patients, food pathogen testing, blood supply screening, and the like, e.g. Mackay, Clin. Microbiol. Infect., 10: 190-212 (2004); Bernard et al., Clinical Chemistry, 48: 1178-1185 (2002).
Suitable nucleic acid amplification methods include both target amplification and signal amplification and may include, but are not limited to, polymerase chain reaction (PCR), ligation chain reaction (LCR, sometimes referred to as oligonucleotide ligase amplification OLA), cycling probe technology (CPT), strand displacement assay (SDA), Rolling circle amplification (RCA), transcription mediated amplification (TMA), nucleic add sequence based amplification (NASBA), and invasive cleavage technology. All of these methods require either two primers or a single primer to hybridize to a target sequence to initiate the amplification. All of these methods require a primer nucleic acid (including nucleic acid analogs) that is hybridized to a target sequence to form a hybridization complex, and an enzyme is added that in some way modifies the primer to form a modified primer. For example, PCR generally requires two primers, dNTPs and a DNA polymerase; LCR requires two primers that adjacently hybridize to the target sequence and a ligase; CPT requires one cleavable primer and a cleaving enzyme; invasive cleavage requires two primers and a cleavage enzyme. etc.
Despite the advances in nucleic acid amplification techniques that are reflected in such widespread applications, there has been limited achievement in performing these techniques in parallel within the same sample, i.e. in multiplexed assays, where multiple target sequences are simultaneously amplified and detected in the same reaction mixture, e.g. Einifro et al., Clin. Microbiol. Rev., 13: 559-570 (2000); Henegarin et al., Biotechniques, 23: 504-511 (1997). When a multiplex assay involves different priming events for different target sequences, the relative efficiency of these events may vary for different targets. This is due to the stability and structural differences between the various primers used. If the rates of product strand renaturation differ for different targets, the extent of competition with priming events will not be the same for all targets. For reactions involving multiple ligation events, such as LCR, there may be small but significant differences in the relative efficiency of ligation events for each target sequence. Since the ligation events are repeated many times, this effect is magnified. For reactions involving reverse transcription (3SR, NASBA) or klenow strand displacement (SDA), the extent of polymerization processivity may differ among different target sequences. For assays involving different replicatable RNA probes, the replication efficiency of each probe is usually not the same, and hence the probes compete unequally in replication reactions catalyzed by Q.beta. replicase. Accordingly, there is a need for amplification methods that are less likely to produce variable and possibly erroneous signal yields in multiplex assays.
Microarray technology has provided an alternative approach for making simultaneous measurements on samples containing multiple polynucleotide analytes, e.g. U.S. Pat. No. 5,700,637, Wang et al., Proc. Nat. Acad. Sci., 99: 15687-15692 (2002); however, such technology has found limited use outside of research laboratories. Furthermore, this technology follows the dominant approach in multiplexed analysis which is to obtain data on every analyte of interest present in the sample. As such, multiplexed assays are primarily concerned with resolving and distinguishing among the plurality of analytes targeted. The number of targets is generally limited in some manner by either the recognition event (e.g. specificity of a probe for a target; the difficulty in designing primer sets that do not interact to negatively effect the amplification) or the detection method (e.g. broad absorption or emission profiles of a chromophore or lumophore limit the number of such labels that can be independently determined). Nonetheless, much work in the field is directed to improving the multiplex capabilities of existing methods by being able to detect or resolve more analytes within a single process.
More recently, several highly multiplexed and ultra-high-throughput genotyping systems have become generally available (Matsuzaki, et al., Nat. Methods 1: 109-111 (2004), Hardenbol, et al., Genome Res. 15: 269-275 (2005), Murray, S. S., et al., Nat. Methods, 1: 113-117 (2004)). However, most of these systems are not flexible due to probe length limitation, and still have limited multiplicity in sample processing due to interprobe hybridization. Therefore, there is a need for developing of sensitive and precise nucleic acid detection methods with high multiplicity.